Thermodynamic Analysis of Weak Protein Interactions Using Sedimentation Equilibrium

Yuri V. Sergeev1, Monika B. Dolinska1, Paul T. Wingfield2

1 National Eye Institute, National Institutes of Health, Bethesda, Maryland, 2 National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 20.13
DOI:  10.1002/0471140864.ps2013s77
Online Posting Date:  August, 2014
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Proteins self‐associate to form dimers and tetramers. Purified proteins are used to study the thermodynamics of protein interactions using the analytical ultracentrifuge. In this approach, monomer‐dimer equilibrium constants are directly measured at various temperatures. Data analysis is used to derive thermodynamic parameters, such as Gibbs free energy, enthalpy, and entropy, which can predict which major forces are involved in protein association. Curr. Protoc. Protein Sci. 77:20.13.1‐20.13.15. © 2014 by John Wiley & Sons, Inc.

Keywords: sedimentation equilibrium; weak protein interaction; thermodynamics

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Table of Contents

  • Introduction
  • Thermodynamics of Protein Association: Theory and Data Analysis
  • Basic Protocol 1: Temperature‐Dependent Sedimentation Equilibrium to Monitor Protein Associate Formation and Determine Dissociation Constants
  • Support Protocol 1: Derivation of Thermodynamic Parameter from Dissociation Constants
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Temperature‐Dependent Sedimentation Equilibrium to Monitor Protein Associate Formation and Determine Dissociation Constants

  • Protein, purified and of known concentration (0.2 µM)
  • Buffer B (see recipe)
  • Buffer A (see recipe)
  • Reductant TCEP (Thermo Scientific)
  • Dialysis membranes Snake or Slide‐A‐Lyzer dialysis kits (Thermo Scientific)
  • Gel filtration column
  • UV‐Vis spectrophotometer (suppliers include Agilent and Beckman)
  • Centrifugation was carried out using a Beckman Coulter Optima XL‐I analytical ultracentrifuge; absorption optics, an An‐60 Ti rotor, and standard double‐sector centerpiece cells are used (Beckman)
  • Beckman XL‐A/ XL‐I data analysis software V6.04
  • Additional reagents and equipment for dialysis ( appendix 3B) or buffer exchange using a desalting column (unit 8.3) and SDS‐PAGE (see the protocol 1Basic Protocol in unit 10.1)
NOTE: The amount of protein used for sedimentation equilibrium depends mainly on the availability of the protein. Typically, 3 to 5 mg is required.NOTE: Always make sure that the solution has reached the required temperature before starting the measurement. Extra time must be allowed for temperature equilibration if the protein solution has been on ice before the experiment.
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Literature Cited

Literature Cited
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